Triglycerides and cholesterol are the main types of fats that are transported in blood, and measurements of triglycerides and cholesterol has been proven to be an effective indicator of potential risk to atherosclerotic cardiovascular disease (CVD).
Triglyceride levels may be elevated in persons with diabetes, kidney, liver or heart diseases, and individuals with elevated triglyceride levels may be at higher risk for heart diseases.
Cholesterol is a sterol lipid essential to the cells of the body and mainly produced by the liver. As cholesterol is hydrophobic it cannot be dissolved and transported in the bloodstream directly, but is transported as part of lipoproteins, which are subdivided into three different classes based on their density. The very-low density lipoproteins (VLDL) are triglyceride-rich lipoproteins which are synthesized in the liver and ultimately converted to low-density lipoproteins (LDL), which transport most of the plasma cholesterol in humans (about 80%). The high-density lipoproteins (HDL) are lipoproteins which are involved in the catabolism of triglyceride-rich lipoproteins and the removal of cholesterol from peripheral tissues and transport to the liver. An inverse relationship between serum HDL levels and risk of CVD has been established. In particular, if the proportion of total serum cholesterol (TC) associated with HDL is low then the risk of CVD is increased.
For clinical analytical purposes in relation with atherosclerotic cardiovascular disease, the levels of the triglycerides (TG), very-low density lipoproteins (VLDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL) are of marked interest, but also the total concentration of cholesterol (TC) is important.
In connection with the introduction of several new drugs, such as statins, for hypercholesterolemia treatment during the end of 1990's, the need for point of care methods, both for screening and monitoring of cholesterol and triglycerides has increased and the clinical demands for precision and accuracy are high with a desirable imprecision of less than around 3%. It is evident that a simple, fast and temperature independent test for quantitative determination of different levels of cholesterol (total, HDL and LDL) and level of triglycerides in blood would be an important aid at doctor's offices.
Both chemical and enzymatic methods for total cholesterol measurement, HDL, LDL and triglycerides are known and measuring of total cholesterol in plasma or serum is performed in central laboratories in hospitals.
The most commonly used chemical method for total cholesterol in plasma or serum is the Liebermann-Burchard reaction, wherein cholesterol reacts as a typical alcohol with strong, concentrated acids producing a coloured substance. Today however mostly enzymatic methods are used. The enzymatic reactions start with a hydrolysis of cholesterol esters to form free cholesterol and the free cholesterol is then oxidized by the enzyme cholesterol oxidase. This enzyme is distinguished by good stability, it is easy to use and it is commercially available.
Another enzyme used in cholesterol determinations is cholesterol dehydrogenase, the use of which is disclosed in an analytical element in the patent publication EP 0 244 825. According to this publication the sample has to be incubated at a specific temperature for a prescribed time. The use of cholesterol dehydrogenase for determination of cholesterol is also disclosed in e.g. the U.S. Pat. Nos. 4,892,816 and 4,181,575. Both patents concern the determination of total cholesterol by wet chemical methods including long incubation times and defined temperatures.
For measurement of HDL, both heterogeneous methods and homogeneous methods are known. In the heterogeneous method LDL and VLDL are precipitated, and HDL aggregates are separated from the supernatant after centrifugation. In the homogeneous methods LDL and VLDL are chemically bound to block the reaction sites thereof from reacting with enzymes for cholesterol determination disclosed above.
Despite the fact that chemical measurement methods are known for total cholesterol measurement, HDL, LDL and triglycerides, there is still no quick, reliable and point of care test system designed for quantitative determination of the concentrations of TC, TG, HDL and LDL in capillary or venous whole blood. CVDs are increasing within today lifestyle and it is important to be able to receive a quick and reliable determination of these analytes.
Determination of blood glucose levels are important in relation to monitoring both diabetes type 1 and type 2 and a number of other metabolic disorders, e.g. metabolic syndrome. Metabolic syndrome is defined as a combination of several medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. The medical disorders defining metabolic syndrome are obesity, raised blood pressure, raised triglyceride value, reduced HDL cholesterol value and raised fasting plasma glucose value (FPG). Blood glucose levels in whole blood are preferably determined by the method disclosed in U.S. Pat. No. 5,278,047 wherein the whole blood is mixed with glucose dehydrogenase, diaphorase, NAD, a hemolysing agent and a redox indicator dye (e.g. MTT) and a colour change, due to the reaction between the glucose in the blood and the reagents, is measured by spectrophotometer. The determination is carried out in a disposable device (microcuvette) including a freeze-dried reagent of the type first disclosed in U.S. Pat. No. 4,088,448. The method of U.S. Pat. No. 5,278,047 is based on the method disclosed in U.S. Pat. Nos. 4,120,755 and 3,964,974 wherein glucose is determined enzymatically using glucose dehydrogenase. In U.S. Pat. No. 4,120,755 a kinetic method for glucose determination in a liquid is disclosed and in U.S. Pat. No. 3,964,974 a method for glucose determination in serum is disclosed.
In order to provide a new, fast and simple method for the determination of analytes for a proper overview of the potential risk of cardiovascular disease and/or metabolic disorders, a disposable device (microcuvette) including a dry reagent of the type first disclosed in the U.S. Pat. No. 4,088,448 was especially studied, as the use of this type of microcuvette offers several advantages.
A microcuvette permits sampling of a liquid, mixing the sample with a suitable reagent, for instance for colour development, in the same vessel as the one used for the subsequent measurement. Furthermore the sampling procedure is simplified, the number of utensils is reduced and in most cases, depending upon the type of analysis, the exactitude of the analysis is considerably improved by making the analysing procedure independent of the operating technique of the operator making the analysis. The procedure is also remarkably fast as it permits the liquid sample to be instantly mixed with the reagent and then permits measurement shortly afterwards, without time consuming intermediary steps.